Collaborative Research: TRTech-PGR: Optimization of Virus-based Delivery of Guide RNAs for Heritable Editing in Maize

CRISPR/CAS的出现使人们能够以前所未有的规模精确编辑基因。目前实现基因编辑全部潜力的一个主要瓶颈是缺乏将基因编辑成分输送到植物中的可靠方法。最广泛使用的将基因编辑成分导入植物细胞的方法是通过转化。然而，许多主要农作物对转化具有顽固性，生产转基因植株费时费力。由于玉米的转化仍然局限于专门的实验室，这是CRISPR/CAS应用于功能基因组研究的主要障碍。在这里，我们将生成资源并开发工具，以简化基因编辑技术在玉米中的应用。鉴于玉米是世界上产量最高的作物，每英亩产生的卡路里比几乎任何其他作物都多，在该项目下开发的工具将对涉及玉米和其他重要单子叶作物的研究产生更广泛的影响。在该项目下产生的所有生物材料和方法将向科学界提供。该项目还将为未被充分代表的少数民族本科生和高中生提供实践研究培训。在植物中开发更新、更有效的基因编辑方法对于进行基因功能研究至关重要。基于CRISPR/CAS的技术的出现和部署使得产生具有高度特异性突变的突变基因型成为可能。到目前为止，大多数基于CRISPR的高效基因编辑方法依赖于传统的转基因方法来传递Cas核酸酶和单引导RNA(SgRNA)成分。虽然能转化玉米自交系的自交系很少，但转化是玉米功能基因组研究的一大瓶颈。此外，传统自交系世代时间较长，需要大量的生长空间。为了克服这些瓶颈，该项目将在一个结构性泛素启动子的控制下培育出高水平表达Cas9核酸酶的B104玉米品系。用于将sgRNAs导入这些表达Cas9的玉米品系的病毒载体将被设计和优化，以诱导体细胞和可遗传的编辑。为了加快玉米基因功能的研究，将建立在结构性泛素和减数分裂特异启动子控制下高水平表达Cas9的快速开花迷你玉米(FFMM)系。这些提供sgRNA的品系和优化的病毒载体将接受体细胞和可遗传编辑的诱导测试。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The advent of CRISPR/Cas has enabled the ability to precisely edit genes at an unprecedented scale. A current major bottleneck in realizing the full potential of gene editing is lack of robust methods for the delivery of gene editing components into plants. The most widely used method for the delivery of gene editing components into plant cells is through transformation. However, many major crops are recalcitrant to transformation and production of transgenic plants is time consuming and labor-intensive. Since transformation of maize is still limited to specialized laboratories, it is a major impediment in application of CRISPR/Cas for functional genomic studies. Here, we will generate resources and develop tools that will ease application of gene editing technology in maize. Given maize is the world’s most produced crop, yields more calories per acre than almost any other crop, the tools developed under this project will have broader impact on studies involving maize and other important monocot crops. All biological materials and the methods generated under this project will be made available to the scientific community. The project will also provide hands-on research training to underrepresented minority undergraduate students and high school students. Developing newer, more efficient gene editing methods in plants is critical to performing gene function studies. The advent and deployment of CRISPR/Cas-based technology has allowed for the generation of mutant genotypes with highly specific mutations. So far, most methods for highly efficient, CRISPR-based gene editing rely on traditional transgenic approaches to deliver the Cas nucleases and single guide RNA (sgRNA) components. Although few maize inbred lines can be transformed, transformation is a major bottleneck in maize functional genomic studies. Furthermore, traditional inbred lines have longer generation times and require a significant amount of growth space. To overcome these bottlenecks, this project will generate B104 maize lines that express high levels of Cas9 nuclease under the control of a constitutive ubiquitin promoter. Viral vectors for delivery of sgRNAs into these Cas9 expressing maize lines will be engineered and optimized for induction of somatic and heritable editing. To accelerate gene function studies in maize, Fast Flowering Mini Maize (FFMM) lines expressing high levels of Cas9 under the control of the constitutive ubiquitin and meiosis-specific promoters will be generated. These lines and optimized viral vectors to deliver sgRNAs will be tested for induction of somatic and heritable editing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.